Project Summary/Abstract: I have completed DVM, MS, and PhD degrees, a surgical residency, and am now a Post-Doctoral Research Fellow in cancer and bone genetics and biology. This K01 award will facilitate my transition into an independent position as a tenure-track clinician-scientist studying bone metastasis and primary bone tumors. My clinical and research training will benefit both human and veterinary clinicians and researchers and promote the development of essential translational and comparative collaborations. Advancement towards independence will occur by many mechanisms during this K01 award. I will obtain knowledge and skills central to this award?s research strategy and my career goals. I will continue to hone previously learned techniques but also receive training in new methods to study cancer biology and genetics, the tumor microenvironment, bone biology, and metastasis. I will receive training in essential non-laboratory research skills such as grantsmanship, scientific writing, responsible conduct of research, and mentoring. Metastatic disease is the leading cause of breast cancer-related deaths and bone metastases occur in ~75% of patients with metastasis. In bone, cancer cells secrete factors that 1) stimulate macrophages (MPs) to differentiate into osteoclasts (OCs) and 2) increase osteoclastic bone resorption (lysis), leading to bone loss, pain, and fracture. Therefore, preventing the establishment of and treating existing bone metastasis is the goal of therapy. Colony stimulating factor 1 receptor (CSF1R) regulates the expression of the transcription factor (TF) PU.1. Our preliminary data demonstrate that the CSF1R/PU.1 axis is a key regulator of 1) tumor-associated MP (TAM) function and 2) normal MP and OC differentiation and function. In addition, several TFs and genes essential for OC differentiation and function have a strong enhancer binding pattern by PU.1 resembling ?superenhancers?. These enhancers are enriched for BET proteins which bind to PU.1 to regulate key osteoclastogenic TFs. The objective of this award is to 1) investigate the role of the CSF1R/PU.1 axis and 2) determine if superenhancers play a role, in breast cancer bone metastasis. Our hypothesis is that the CSF1R/PU.1 axis is essential for bone metastasis and this occurs in the context of superenhancers. We will initially assess the effects of MP-specific deletion of PU.1 on breast cancer bone metastasis and determine genes regulated by PU.1 in both primary tumor MPs and bone metastasis MPs and OCs (Aim 1). We will then evaluate if BET inhibition can reduce the development of, and growth of established, bone metastasis and determine if increasing its specificity to specifically target MPs and OCs will increase its therapeutic efficacy (Aim 2). Lastly, we will evaluate if combination therapy with the most efficacious BET inhibitor preparation from Aim 2 and a CSF1R inhibitor targeting the CSF1R/PU.1 axis is more efficacious against the development and growth of bone metastasis when compared to either agent alone (Aims 3 and 4A- B). Findings could have a major impact on treatment, quality of life, and survival of bone metastasis patients.